This research aims to construct a lentiviral expression vector carrying the extracelluar domain (ED) of human hepatocyte growth factor receptor (C-Met), and to express it in transfected 293T cells. The extracellular domain of C-Met was amplified by RT-PCR, ligated with lentiviral expression vector p RRL-CMV-ED, and then expressed in 293T cell line. The expressed protein was purified and identified by RT-PCR and Western blot. The enzyme digestion and sequence analysis showed that the lentiviral expression vector p RRL-CMV-ED was constructed correctly. The size of amplified genes was about 2 700 bp. The purified protein with Ni-affinity column was about 105 kD analyzed by SDS-PAGE. The Western blot and ELISA results showed that the expressed protein which could bind to HGF specifically was the extracelluar domain of human hepatocyte growth factor receptor. This research may lay a foundation for further study of anti-C-MET monoclonal antibody and neutralizing antibody.
ObjectiveTo establish 16HBE cell lines stably expressing glutathione S-transferase mu 5 (GSTM5) gene, and explore the mechanism of GSTM5 nuclear translocation. MethodsRecombinant lentiviral expression vector containing GSTM5 gene was constructed and lentivirus was produced. After lentivirus infection of 16HBE cells, 16HBE-GSTM5 cell lines were obtained by screening with puromycin. Expression of GSTM5 in different cells was examined by RT-qPCR and Western blot. The nuclear translocation of GSTM5 was observed by confocal laser scanning microscope, after the 16HBE-GSTM5 cell lines were treated with tumor necrosis factor-α (TNF-α; 10 ng/ml) for 0.5 hour. ResultsLentiviral expression plasmids, PLVX-puro-3*flag-SBP-GSTM5-C and PLVX-puro-GSTM5-SBP-3*flag-N, were constructed and lentiviral particles were successfully packed. After infected with lentivirus and screened by puromycin, two cell lines, 16HBE-GSTM5-SBP-3*flag-N and 16HBE-3*flag-SBP-GSTM5-C, were obtained. GSTM5 expression in these two cell lines was significantly higher compared with the control group and parental cells. After treated with TNF-α for 0.5 hour, the nuclear translocation of GSTM5 in 16HBE-GSTM5-SBP-3*flag-N was much more obviously than that in 16HBE-3*flag-SBP-GSTM5-C. ConclusionThe N-terminal region of GSTM5 is critical for nuclear translocation induced by TNF-α, which is mediated by a novel and non-classical nuclear localization signal.